Method and composition for regulating apoptosis

ABSTRACT

Methods and compositions for preventing or inhibiting apoptosis are provided by this invention. The methods require introducing into a cell which may undergo apoptosis a nucleic acid molecule coding for a gene product having crmA biological activity or a crmA polypeptide. This invention also provides compositions and methods for maintaining T cell viability in a subject infected with the human immunodeficiency virus (HIV), by administering to the subject an effective amount of a nucleic acid molecule coding for a gene product having crmA biological activity or the gene product itself.

This invention was made in part with Government support under Grant No.CA61348 awarded from the National Institute of Health. The U.S.government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to methods for regulating apoptosis in apopulation of cells as well as compositions useful to regulateapoptosis.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death (PCD) is a type of cell death thatis fundamentally distinct from degenerative death or necrosis. It is anactive process of gene-directed cellular self-destruction which in someinstances, serves a biologically meaningful homeostatic function. Thiscan be contrasted to necrosis which is cell death occurring as theresult of severe injurious changes in the environment of infected cells.For a general review of apoptosis, see Tomei, L. D. and Cope, F. O.Asoptosis: The Molecular Basis of Cell Death (1991) Cold Spring HarborPress, N.Y.; Tomei, L. D. and Cope, F. O. Apoptosis II: The MolecularBasis of Apoptosis in Disease (1994) Cold Spring Harbor Press, N.Y.; andDuvall and Wyllie (1986) Immun. Today 7(4):115-119.

Morphologically, apoptosis is characterized by the rapid condensation ofthe cell with preservation of membranes. Synchronistically with thecompaction of chromatin, several biochemical changes occur in the cell.Nuclear DNA is cleaved at the linker regions between nucleosomes toproduce fragments which are easily demonstrated by agarose gelelectrophoresis wherein a characteristic ladder develops.

Apoptosis has been linked to many biological processes, includingembryogenesis, development of the immune system, elimination ofvirus-infected cells, and the maintenance of tissue homeostasis.Apoptosis also occurs as a result of human immunodeficiency virus (HIV)infection of CD4⁺ T lymphocytes (T cells). Indeed, one of the majorcharacteristics of AIDS is the gradual depletion of CD4⁺ T lymphocytesduring the development of the disease. Several mechanisms, includingapoptosis, have been suggested to be responsible for the CD4 depletion.It is speculated that apoptotic mechanisms might be mediated eitherdirectly or by the virus replication as a consequence of the HIVenvelope gene expression, or indirectly by priming uninfected cells toapoptosis when triggered by different agents.

The depletion of CD4⁺ T cells results in the impairment of the cellularimmune response. It has been proposed that an inappropriateactivation-induced T cell PCD causes the functional and numericalabnormalities of T_(H) cells from HIV-infected patients, that leads tothe near collapse of the patient's immune system.

Therefore, it is advantageous to block apoptosis and the ensuingdepletion of T cells. Accordingly, a need exists to maintain T cellfunction and viability in HIV infected individuals and to providesystems to screen for new drugs that may assist in maintaining thecellular immune response. This invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

This invention provides compositions and methods for preventing orinhibiting apoptosis in a suitable cell by introducing into the cell anucleic acid molecule coding for a gene product having crmA biologicalactivity or alternatively, the crmA gene product.

Also provided by this invention are compositions and methods forpreventing or inhibiting induced apoptosis in a suitable cell byintroducing into the cell a nucleic acid molecule coding for a geneproduct having cytokine response modifier crmA biological activity orthe gene product so that induced apoptosis is prevented or inhibited.

Further provided by this invention are compositions and methods formaintaining T cell viability in a subject infected with or susceptibleto infection with the human immunodeficiency virus by administering tothe subject an effective amount of a nucleic acid molecule coding for agene product having crmA biological activity or the crmA gene product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows TNF and anti-Fas+CHX induce apoptosis in MCF7 cells. MCF7cells were treated with TNF or anti-Fas+CHX as described in theexperimental section below. Upper row: Nuclei of untreated, TNF treated,or anti-Fas+CHX treated MCF7 cells stained with propidium iodide andvisualized by laser-scanning confocal microscopy. Arrows indicateexamples of apoptotic nuclei. Lower row: Transmission electronmicroscopy of untreated, TNF treated or anti-Fas+CHX treated MCF7 cells.FIG. 1B shows that anti-Fas antibody induces apoptosis in BJAB cells.BJAB cells were treated with anti-Fas antibody as described below. TheFigure shows fluorescence microscopy of untreated or anti-Fas treatedBJAB cells stained with acridine orange. Arrows indicate examples ofapoptotic nuclei. Inset: Laser scanning confocal microscopy of untreatedor anti-Fas treated BJAB cells stained with acridine orange.

FIG. 2 shows inhibition of apoptosis by crmA in pooled populations oftransfectants. Pooled populations of the vector or crmA transfectedcells indicated were analyzed for sensitivity to TNF- or Fas-mediatedapoptosis.

FIG. 3A shows inhibition of TNF and anti-Fas+CHX induced apoptosis ofMCF7 cells by crmA. Top: Sensitivity of MCF7 vector transfected clones(V1-V5) or crmA transfected clones (crmA1 to crmA5) to TNF andanti-Fas+CHX induced cell death assessed by propidium iodine apoptosisassay. Insert: Sensitivity of selected clones to TNF and anti-Fas+CHXinduced cell death by MTT conversion assay.

FIG. 3B top: Northern analysis of corresponding cell lines to detectcrmA transcript. FIG. 3B bottom: Northern analysis to detect β-actintranscript loading of RNA.

FIG. 3C shows crmA-mediated inhibition of apoptosis induced byincreasing doses of anti-Fas antibody.

FIG. 3D shows crmA inhibits apoptosis induced by increasing doses ofanti-Fas antibody. The acridine orange apoptosis assay was used toanalyze the sensitivity of vector transfected (BJAB V1) orcrmA-transfected (BJAB crmA2, BJAB crmA3) clones to apoptosis induced bythe indicated doses of anti-Fas antibody.

FIG. 4A shows inhibition of anti-Fas induced apoptosis in BJAB cells bycrmA. Top: Analysis of sensitivity of BJAB vector transfected clones(V1-V6) or crmA transfected clones (crmA1-crmA10) to anti-Fas inducedapoptosis. Cell death was assessed by acridine orange apoptosis assay.Insert: Sensitivity of selected clones to anti-Fas induced cell deathassessed by MTT conversion assay.

FIG. 4B top: Northern analysis of corresponding cell lines for detectionof expression of crmA transcript. FIG. 4B bottom: Northern analysis todetect β-actin transcript to assess loading RNA.

FIG. 4C shows crmA inhibits apoptosis by increasing doses of anti-Fasantibody. The acridine orange apoptosis assay was used to analyze thesensitivity of vector transfected (BJAB V1) or crmA-transfected (BJABcrmA2, BJAB crmA3) clones to apoptosis induced by the indicated doses ofanti-Fas antibody.

FIGS. 5A through 5C show the nucleic acid sequence and correspondingamino acid sequence of the cowpox crmA gene and gene product. (Seq. ID.Nos. 3 and 4, respectively.)

DETAILED DESCRIPTION OF THE INVENTION

As is known to those of skill in the art, apoptosis is an active processof gene-directed cellular self-destruction. This invention providescompositions and methods for preventing or inhibiting apoptosis in asuitable cell or a population of suitable cells by introducing into thecell or cells an effective amount of a nucleic acid molecule coding fora gene product having crmA biological activity. The method also may bepracticed using the gene product itself. It is important to note thatthe method of this invention inhibits apoptosis even in the presence ofapoptotic-inducing agents, such as receptor ligands, e.g., anti-TCR,tumor necrosis factor (TNF), HIV, SIV or anti-Fas antibody. Accordingly,this method provides an improvement over prior art methods whereinapoptosis can be inhibited by interfering with the induction pathway atthe level of ligand induction, such as by providing antibodies oranti-ligand antibodies to interfere with the binding of the ligand toits cell surface receptor. However, this invention can be combined withthe use of such prior art methods to inhibit apoptosis.

The terms "preventing" or "inhibiting" are intended to mean a reductionin cell death or a prolongation in the survival time of the cell. Theyalso are intended to mean a diminution in the appearance or a delay inthe appearance of morphological and/or biochemical changes normallyassociated with apoptosis. Thus, this invention provides compositionsand methods to increase survival time and/or survival rate of a cell orpopulation of cells which, absent the use of the method, would normallybe expected to die. Accordingly, it also provides compositions andmethods to prevent or treat diseases or pathological conditionsassociated with unwanted cell death in a subject.

Suitable cells or "target cells" for the practice of this methodinclude, but are not limited to, cells that are induced to PCD by anendogenous agent such as HIV, anti-TCR antibody, TNF and anti-Fasantibody. In one embodiment, these cells constitutively and induciblyexpress receptors for either or both of the cytokine tumor necrosisfactor (TNF) or the cell death transducing receptor Fas or TCR and whichhave been activated by their respective ligand. Recently, three separategroups have reported that Fas-induced apoptosis is involved in T celldeath. Specifically, one group has shown that the Fas receptor, whichcan transduce a potent apoptotic signal when ligated, is rapidlyexpressed following activation on T cell hybridomas. It was suggestedthat the Fas receptor-ligand interaction induces cell death in acell-autonomous manner. See Dhein et al. (1995) Nature 373:438-441;Brunner et al. (1995) Nature 373:441-444; and Ju et al. (1995) Nature373:444-448.

For the purpose of illustration only, examples of suitable cells are Tlymphocytes (T cells) (e.g., TCR⁺, CD4⁺ and CD8⁺ T cells) leukocytes andmixed leukocyte cultures (MLC), B lymphoma cells (e.g., A202J (ATCC)),bone marrow cells, endothelial cells, breast carcinoma cells, fibroblastcells, epithelial tumor cells (see Spriggs, D. R. et al. (1988) J. Clin.Inves. 81:455-460) and monocytes. Fas and TNF receptor expression alsohas been identified on numerous tissues, see for exampleWatanabe-Fukunaga et al. (1992) J. Immun. 148:1274-1279 and Owen-Schaub,L. B. et al. (1994) Cancer Res. 54:1580-1586; Dhein et al. (1995) Nature373:438-441; Brunner et al. (1995) Nature 373:441-444; and Ju et al.(1995) Nature 373:444-448. Assays for identifying additional "suitable"cells sensitive to induction or activation, e.g., TCR-, TNF- orFas-related apoptosis, are well known to those of skill in the art. (Seefor example, Opipari, et al. J. Biol. Chem. (1992) 267:12424-12427;Yonehara et al. J. Exp. Med. (1989) 169:1747-1756; Dhein et al. (1995)supra; Brunner et al. (1995) supra and Ju et al. (1995) supra) However,this method is particularly suitable for use with TCR⁺, CD8⁺ or CD4⁺ Tcells or tissues that harbor the simian immunodeficiency virus (SIV) oralternatively, the human immunodeficiency virus (HIV). The cells can bemammalian cells or animal cells, such as guinea pig cells, rabbit cells,simian cells, mouse cells, rat cells, or human cells. They can becontinuously cultured or isolated from an animal or human. In a separateembodiment of this invention, neurological cells are specificallyexcluded.

This invention is based on Applicants' finding that the cowpox viruscrmA gene product is an exceptionally potent inhibitor of apoptosisinduced by binding of a cell surface receptor to its ligand, e.g., TCRligand, HIV, Fas or TNF. In one embodiment which utilized TNF- and Fas-pathways; it is capable of blocking the cell death program even atpharmacological doses of the death stimulus. crmA is a cowpox virus genewhich encodes a protease inhibitor of the serpin family. The nucleicacid and corresponding amino acid sequences of crmA have been reported(Pickup et al., Proc. Natl. Acad. Sci. (1986) 83:7698-7702) and areshown in FIG. 5.

The only reported target for the crmA protein is the cysteine proteaseinterleukin-1β converting enzyme (ICE). However, Applicants have foundthat crmA is an exceptionally potent inhibitor of apoptosis. Therefore,an important new function for crmA is the prevention or inhibition ofligand-induced or cytokine-induced apoptosis. Further, the data suggestthat a protease, either ICE or a related crmA-inhibitable protein, is acomponent of the Fas- and TNF-induced cell death pathways. Thus, thisinvention provides: compositions and methods for preventing orinhibiting ligand-induced or cytokine-induced apoptosis; an assay fordetermining drugs or agents which facilitate or prevent or inhibitapoptosis; an assay for drugs to treat or ameliorate the symptomsassociated with a disease or pathological conditions that occur as aresult of apoptosis (such as AIDS); an assay for detecting the proteaseinvolved in the Fas- and TNF- induced cell death pathways, as well theproteases discovered using this method.

The crmA gene or nucleic acid can be isolated from natural or nativesources as described in Pickup et al. (1986) supra. The term "native"refers to the form of a nucleic acid, protein, polypeptide, antibody ora fragment thereof that is isolated from nature or which is without anintentional amino acid substitution. As used herein, "nucleic acid" and"gene" are synonymous and shall mean single and double stranded genomicDNA, cDNA, mRNA and cRNA. "Isolated" when used to describe the state ofthe nucleic acids or proteins, denotes the nucleic acids or proteinsfree of at least a portion of the molecules associated with or occurringwith nucleic acids in their native environment.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989) and the various references cited therein. This reference and thecited publications are expressly incorporated by reference into thisspecification.

The DNA sequence provided in FIG. 5 can be duplicated using a DNAsequencer and methods well known to those of skill in the art. Forexample, the sequence can be chemically replicated using PCR(Perkin-Elmer) which in combination with the synthesis ofoligonucleotides, allows easy reproduction of DNA sequences. A DNAsegment of up to approximately 6000 base pairs in length can beamplified exponentially starting from as little as a single gene copy bymeans of PCR. In this technique, a denatured DNA sample is incubatedwith two oligonucleotide primers that direct the DNApolymerase-dependent synthesis of new complementary strands. Multiplecycles of synthesis each afford an approximate doubling of the amount oftarget sequence. Each cycle is controlled by varying the temperature topermit denaturation of the DNA strands, annealing the primers, andsynthesizing new DNA strands. The use of a thermostable DNA polymeraseeliminates the necessity of adding new enzyme for each cycle, thuspermitting fully automated DNA amplification. Twenty-five amplificationcycles increase the amount of target sequence by approximately 10⁶-fold. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065, and 4,683,202.

The nucleic acid can be duplicated using a host-vector system andtraditional cloning techniques with appropriate replication vectors. A"host-vector system" refers to host cells which have been transfectedwith appropriate vectors using recombinant DNA techniques. The vectorsand methods disclosed herein are suitable for use in host cells over awide range of eucaryotic organisms. This invention also encompasses thecells transformed with the novel replication and expression vectorsdescribed herein.

Indeed, the crmA gene can be duplicated in many replication vectors suchas the vaccinia virus as described in Pickup et al. (1986) supra, andisolated using methods described in Sambrook et al. (1989) supra.

The crmA gene made and isolated using the above methods can be directlyinserted into an expression vector, such pcDNA3 (Invitrogen) andinserted into a suitable animal or mammalian cell such as a guinea pigcell, a rabbit cell, a simian cell, a mouse, a rat or a human cell.

In the practice of one embodiment of this invention, the crmA nucleicacid molecule is introduced into the cell and expressed and cell deathis aborted. A variety of different gene transfer approaches areavailable to deliver the crmA gene into a target cell, cells or tissues.Among these are several non-viral vectors, including DNA/liposomecomplexes, and targeted viral protein DNA complexes. To enhance deliveryto a cell, the nucleic acid or proteins of this invention can beconjugated to antibodies or binding fragments thereof which bind cellsurface antigens, e.g., TCR, CD3 or CD4. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. This invention also provides the targeting complexes foruse in the methods disclosed herein.

The crmA nucleic acid also can be incorporated into a "heterologous DNA"or "expression vector" for the practice of this invention. The term"heterologous DNA" is intended to encompass a DNA polymer such as viralvector DNA, plasmid vector DNA or cosmid vector DNA. Prior to insertioninto the vector, it is in the form of a separate fragment, or as acomponent of a larger DNA construct, which has been derived from DNAisolated at least once in substantially pure form, i.e., free ofcontaminating endogenous materials and in a quantity or concentrationenabling identification, manipulation, and recovery of the segment andits component nucleotide sequences by standard biochemical methods, forexample, using a cloning vector. As used herein, "recombinant" isintended to mean that a particular DNA sequence is the product ofvarious combination of cloning, restriction, and ligation stepsresulting in a construct having a sequence distinguishable fromhomologous sequences found in natural systems. Recombinant sequences canbe assembled from cloned fragments and short oligonucleotides linkers,or from a series of oligonucleotides.

As noted above, one means to introduce the nucleic acid into the cell ofinterest is by the use of a recombinant expression vector. "Recombinantexpression vector" includes vectors which are capable of expressing DNAsequences contained therein, where such sequences are operatively linkedto other sequences capable of effecting their expression. It is implied,although not always explicitly stated, that these expression vectorsmust be replicable in the host organisms either as episomes or as anintegral part of the chromosomal DNA. In sum, "expression vector" isgiven a functional definition, and any DNA sequence which is capable ofeffecting expression of a specified DNA sequence disposed therein isincluded in this term as it is applied to the specified sequence.Suitable expression vectors include viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids andothers. Adenoviral vectors are a particularly effective means forintroducing genes into tissues in vivo because of their high level ofexpression and efficient transformation of cells both in vitro and invivo.

Replication-incompetent retroviral vectors also can be used with thisinvention. As used herein, the term "retroviral" includes, but is notlimited to, a vector or delivery vehicle having the ability toselectively target and introduce the coding sequence into dividingcells, such as the mouse molony-leukemia virus. As used herein, theterms "replication-incompetent" is defined as the inability to produceviral proteins, precluding spread of the vector in the infected hostcell. As should be understood by those of skill in the art, crmA nucleicacid will be ribonucleic acid (RNA) for introduction with a retroviralvector.

Another example of a replication-incompetent retroviral vector is LNL6(Miller, A. D. et al., BioTechnicues 7:980-990 (1989)). The methodologyof using replication-incompetent retroviruses for retroviral-mediatedgene transfer of gene markers is well established (Correll, P. H. etal., (1989) PNAS USA 86:8912; Bordignon, C. et al., PNAS USA (1989)86:6748-52; Culver, K. et al., (1991) PNAS USA 88:3155; Rill, D. R. etal., (1990) Blood 79(10):2694-700). Clinical investigations have shownthat there are few or no adverse effects associated with these viralvectors (Anderson, (1992) Science 256:808-13).

In one embodiment of this invention, the expression vector is to bespecifically targeted to T cells. For these methods, it intended thatthe crmA DNA be operatively linked to a promoter that is highly activein T cells. Such promoters include, but are not limited to: IFN-τ; IL-2;IL-3; IL-4; IL-5; IL-9; IL-10; TFN-β; GM-CSF; CD4, CD8 and the IL-2promoter.

Although the method is preferably practiced with the crmA gene, itshould be apparent to those of skill in the art that the polypeptideproduct of the crmA gene and its biological equivalents are useful inthe methods of this invention. The crmA gene product is a 38 kDa proteinand is known to be a specific inhibitor of IL-1β. It can be purifiedfrom natural sources as described in Ray, C. A. (1992) Cell 69:597-604or produced recombinantly using the expression vectors described abovein a host-vector system such as described in Pickup et al. (1986) supra,Ray et al. (1992) supra and Moss, B. ed. (1990) Virology, pp:2079-2112,Raven Press, N.Y. The protein is used in substantially pure form. By"substantially pure," it is meant that the protein is substantially freeof other biochemical moieties with which it is normally associated innature. The proteins also can be produced using the sequence provided inFIG. 5 and methods well known to those of skill in the art.

Accordingly, this invention also provides a crmA polypeptide, protein, abiological equivalent thereof and fusion proteins containing these, foruse in the methods described herein. The polypeptides or proteins can beconjugated to targeting antibodies, such as anti-CD3 or anti-CD4 fortargeted delivery to T cells.

A "biological equivalent" is intended to mean any fragment of thenucleic acid or protein, a mimetic (protein and non-protein mimetic)also having the ability to inhibit apoptosis using the assay systemdescribed and exemplified herein. For example, purified crmA polypeptidecan be contacted with a suitable cell as described above and under suchconditions that apoptosis is inhibited. It is understood that limitedmodifications can be made to the primary sequence of the crmA sequenceas shown in FIG. 5 and used in this invention without destroying itsbiological function, and that only a portion of the entire primarystructure may be required in order to effect biological activity.Examples of such biological equivalent fragments include, but are notlimited to the 5.2 kb EcoRI fragment or the 2.7 kb BglII fragment andthe polypeptides encoded by these nucleic acid molecules as described inPickup et al. (1986) supra. It is further understood that minormodifications of the primary amino acid sequence may result in proteinswhich have substantially equivalent or enhanced function as compared tothe molecule within the vector. These modifications may be deliberate,as through site-directed mutagenesis, or may be accidental such asthrough mutation in hosts. All of these modifications are included aslong as the ability to inhibit apoptosis is retained.

This method can be practiced in vitro, ex vivo or in vivo. When themethod is practiced in vitro, the expression vector, protein orpolypeptide can be added to the cells in culture or added to apharmaceutically acceptable carrier as defined below. In addition, theexpression vector or crmA DNA can be inserted into the target cell usingwell known techniques such as transfection, electroporation ormicroinjection.

More specifically, the in vitro method comprises providing cell culturesor tissue cultures having either a cell surface receptor that mediatesapoptosis such as a TCR, the TNF receptor or the Fas receptor. The cellsare cultured under conditions (temperature, growth or culture medium andgas (CO₂)) and for an appropriate amount of time to attain exponentialproliferation without density dependent constraints. The cells are thenexposed to preliminary conditions necessary for apoptosis, for examplean effective amount of an inducing agent, e.g., a TCR ligand, HIV, SIV,TNF, or a Fas ligand such as an anti-Fas antibody is added to theculture. Anti-Fas antibodies and mitogens (ConA) are well known to thoseof skill in the art. (Itoh, N. et al. (1991) Cell 66:233-243 andYonehara et al. (1989) J. Exp. Med. (1989) 169:1747-1756). These cellsare now "induced" to apoptosis. The cells are again cultured undersuitable temperature and time conditions. In one embodiment, HIV or SIVis added to the culture. In other embodiments, a drug or agent to betested is added in varying concentrations at a time that is simultaneouswith, prior to, or after the inducing agent.

The crmA nucleic acid or protein is then added to the culture in aneffective amount and the cells are cultured under suitable temperatureand time conditions to inhibit apoptosis. The crmA nucleic acid orprotein can be added prior to, simultaneously with, or after, theinducing agent. The cells are assayed for apoptotic activity usingmethods well known to those of skill in the art and described herein. Itis apparent to those of skill in the art that two separate culture ofcells must be treated and maintained as the test population. One ismaintained without receiving an inducing agent to determine backgroundrelease and the second without receiving the agent to be tested. Thesecond population of cells acts as a control.

The use of the compositions and methods in vitro provides a powerfulbioassay for screening for drugs which are agonists or antagonists ofcrmA function in these cells. Thus, one can screen for drugs havingsimilar or enhanced ability to prevent or inhibit apoptosis. It also isuseful to assay for drugs having the ability to inhibit HIV infectionand replication, since the CD4⁺ cell will not die as a result of theconcurrent viral infection. One of skill in the art can determine whenthe method has been successfully performed by noting the absence ofapoptotic morphological changes or more simply, by the absence of celldeath. The in vitro method further provides an assay to determine if themethod of this invention is useful to treat a subject's pathologicalcondition or disease that has been linked to apoptotic cell death in theindividual.

For example, a T cell hybridoma cell line such as Jurkat can be stablytransfected with the crmA expression construct or vector alone andclonal cell lines derived. Transfection of Jurkat cell byelectroporation can be performed as described in Laherty et al. J. Biol.Chem. (1993) 263:5032-5039. crmA-expressing and vector-transfectedcontrol cells are ⁵¹ Cr-labeled and plated (5×10⁵ /ml) on untreated oranti-CD3 (available from the cell line 145-2C11 (ATCC)) treated tissueculture plastic plates. Cells cultured on uncoated cells are used todetermine background release. The percentage cell death will bedetermined at various times after culture by the formula: c.p.m.released from the experimental group minus c.p.m. of background releasedivided by c.p.m. released by 0.5% Triton X-100 (complete lysis)--c.p.m.of background release.

A substantial decrease in percent cell death induced by plating cells onimmobilized anti-CD3 monoclonal antibody is an indication that crmAinhibits T cell receptor-induced death. Using the method describedabove, various agents can be tested for their ability to inhibit orprevent apoptosis.

In a separate embodiment, the T cell line designated CEM (ATCC) isobtained and used because it has been shown to undergo PCD uponinfection with HIV. CEM cells are transfected by electroporation withthe crmA expression construct and vector alone as control. Clonal linesare derived and infected at various multiplicity of infection ratioswith HIV. Cytopathic effect is assayed by microscopic observation andapoptosis quantitated following propidium iodine staining. Using themethod described above, various agents can be tested for their abilityto inhibit or prevent apoptosis.

When the method is practiced in vivo in a human patient, it isunnecessary to provide the inducing agent since it is provided by thepatient's immune system. However, when practiced in an experimentalanimal model, it can be necessary to provide an effective amount of theinducing agent in a pharmaceutically acceptable carrier prior toadministration of the crmA product, to induce apoptosis. When the methodis practiced in vivo, the carrying vector, crmA polypeptide, polypeptideequivalent, or crmA expression vector can be added to a pharmaceuticallyacceptable carrier and systemically administered to the subject, such asa human patient or an animal such as a mouse, a guinea pig, a simian, arabbit or a rat. Alternatively, it can be directly infused into the cellby microinjection. A fusion protein also can be constructed comprisingthe protease-inhibitory region of the crmA, diphtheria toxin and aT-cell specific ligand for targeting to a T cell. Such T cell specificligands include, but are not limited to anti-CD3, anti-CD4, anti-CD28and anti-IL-1 antibody protein.

Acceptable "pharmaceutical carriers" are well known to those of skill inthe art and can include, but not be limited to any of the standardpharmaceutical carriers, such as phosphate buffered saline, water andemulsions, such as oil/water emulsions and various types of wettingagents.

When practiced in vivo, the compositions and methods are particularlyuseful for maintaining T cell viability and function in a subject or anindividual suffering from or predisposed to suffer from abnormallymphocyte death. When the animal is an experimental animal such as asimian (using SIV), this method provides a powerful assay to screen fornew drugs that may be used alone or in combination with this inventionto ameliorate or reduce the symptoms and opportunistic infectionsassociated with HIV infection or AIDS.

This invention also is particularly useful to ward off lymphocyte deathor immunosuppression in AIDS patients. By preventing or inhibitingapoptosis, not only is cell death prevented but functionality, e.g.,immuno-proliferative capacity, is restored to the cell and a responsiveimmune system is retained or regained. Accordingly, the compositions andmethods of this invention are suitably combined with compositions andmethods which prevent or inhibit HIV infectivity and replication.

The method can also be practiced ex vivo using a modification of themethod described in Lum et al. (1993) Bone Marrow Transplantation12:565-571. Generally, a sample of cells such as bone marrow cells orMLC can be removed from a subject or animal using methods well known tothose of skill in the art. An effective amount of crmA nucleic acid isadded to the cells and the cells are cultured under conditions thatfavor internalization of the nucleic acid by the cells. The transformedcells are then returned or reintroduced to the same subject or animal(autologous) or one of the same species (allogeneic) in an effectiveamount and in combination with appropriate pharmaceutical compositionsand carriers.

As used herein, the term "administering" for in vivo and ex vivopurposes means providing the subject with an effective amount of thenucleic acid molecule or polypeptide effective to prevent or inhibitapoptosis of the target cell. Methods of administering pharmaceuticalcompositions are well known to those of skill in the art and include,but are not limited to, microinjection, intravenous or parenteraladministration. The compositions are intended for topical, oral, orlocal administration as well as intravenously, subcutaneously, orintramuscularly. Administration can be effected continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with the vectorused for therapy, the polypeptide or protein used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Single or multiple administrations can be carried outwith the dose level and pattern being selected by the treatingphysician. For example, the compositions can be administered prior to asubject already suffering from a disease or condition that is linked toapoptosis. In this situation, an effective "therapeutic amount" of thecomposition is administered to prevent or at least partially arrestapoptosis and the accompanying pathology such as immunosuppression inHIV infected individuals.

However, the compositions can be administered to subjects or individualssusceptible to or at risk of developing apoptosis-related disease toprevent pathological cell death. In one embodiment, the composition canbe administered to a subject susceptible to HIV-related lymphocytedisfunction to maintain lymphocyte cell function and viability. In thesemethods a "prophylactically effective amount" of the composition isadministered to maintain cellular viability and function at a level nearto the pre-infection level.

It should be understood that by preventing or inhibiting unwanted celldeath in a subject or individual, the compositions and methods of thisinvention also provide methods for treating, preventing or amelioratingthe symptoms associated with a disease characterized by apoptosis ofcells. Such diseases include but are not limited to AIDS, acute andchronic inflammatory disease, leukemia, myocardial infarction, stroke,traumatic brain injury, neural and muscular degenerative diseases,aging, tumor induced-cachexia and hair loss.

This invention also provides vector and protein compositions useful forthe preparation of medicaments which can be used for preventing orinhibiting apoptosis, maintaining cellular function and viability in asuitable cell or for the treatment of a disease characterized by theunwanted death of target cells.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand the following examples are intended to illustrate and not limit thescope of the invention. Other aspects, advantages and modificationswithin the scope of the invention will be apparent to those skilled inthe art to which the invention pertains.

Experiment I

Analysis of Apoptosis--Apoptosis was assessed by the use of fluorescentDNA-staining dyes to reveal nuclear morphology and by transmissionelectron microscopy. For propidium iodide staining, MCF7 cells weregrown on 22 mm² No. 1 glass coverslips (Corning) placed in 35 mm wellsof a 6-well culture dish (Costar). Following treatment with TNF,anti-Fas cycloheximide (CHX), or no treatment, medium was removed andthe wells were rinsed twice with phosphate buffered saline (PBS), fixedin 100% methanol at -20° C. for 10 minutes, washed three times with PBS,and stained at room temperature for 10 minutes in a 100 μg/ml solutionor propidium iodide (Sigma) made in PBS. The coverslips were then washedthree times with PBS, blotted dry and mounted onto glass slides usingVectashield mounting medium for fluorescence (Vector Laboratories). BJABcells were stained using acridine orange (SIGMA) by preparing a wetmount of 30 μl of a cell suspension at a density of approximately 3×10⁵cell/ml mixed with 5 μl of a 100 μg/ml acridine orange solution made inPBS. Both propidium iodide-stained MCF7 and acridine orange-stained BJABnuclei were visualized by fluorescence microscopy using a FITC rangebarrier filter cube. Laser-scanning confocal microscopy was performedusing the Bio-Rad MRC 600 confocal microscope and digitized imagesobtained were artificially colorized.

For electron microscopy, cells were fixed and processed as per standardelectron microscopy procedures.

Experiment II

Quantitative Apoptosis Assays--MCF7 cells or derived transfectants wereplated at a concentration of 2.5×10⁵ cells/well onto glass coverslips.Two days later, after the cells had adhered and spread, TNF oranti-Fas+CHK were added. TNF was added at a final concentration of 20ng/ml, anti-Fas at 25 ng/ml, and CHX (Sigma) at 10 μg/ml. After 22 hoursfor the TNF treated samples or after 18 hours for the anti-Fas+CHXtreated samples, cells were fixed, stained with propidium iodide andmounted as described above. Apoptotic and non-apoptotic cells werequantitated based on nuclear morphology using fluorescence microscopyand the percentage of non-apoptotic cells was calculated. A minimum of100 cells was counted for each sample, and each experiment was done atleast in duplicate. Since a small fraction of cells in any normallygrowing cell culture is undergoing apoptosis, spontaneous apoptosis inuntreated or CHX alone treated samples was also quantitated. Thepercentage of non-apoptotic cells in the TNF or anti-Fas+CHX treatedsamples was then normalized by correcting for the frequency ofspontaneous apoptosis in the untreated or CHX alone samples,respectively.

BJAB cells were grown at 3×10⁵ cells/ml and treated with anti-Fasantibody at a concentration of 250 ng/ml (unless indicated otherwise)for 18 hours after which an aliquot was stained with acridine orange asdescribed above. Apoptotic cells and non-apoptotic cells werequantitated and normalized to untreated samples. Assays were done atleast in duplicate.

Secondary assays of cell death used were the MTT conversion assay (asdescribed in Opipari, A. W. et al. J. Biol. Chem. (1992)267:12424-12427) and crystal violet staining and were done as describedin Tartaglia, L. A. et al. (1993) Cell 74:845-853.

Plasmids, Transfections and Selection of Stably Transfected Lines--ThecrmA gene as shown FIG. 5 was cloned into the pcDNA3 (Invitrogen)mammalian expression vector. The crmA gene was obtained from Dr. DavidPickup (Duke University) and used as a template in a PCR reaction usingcustom oligonucleotide primers with built-in restriction enzyme sites toamplify the entire coding sequence. The sequence of the primers are:

crmA/5'/R1

5' CAC CGG AAT TCC ACC ATG GAT ATC TTC AGG GAA ATC G

(Seq. ID. No. 1)

crmA/3'/XbaI

5' GCT CTA GAC TCG AGT TAA TTA GTT GTT GGA GAG CAA TAT C

(Seq. ID. No. 2)

This PCR fragment was digested with EcoR1 and Xbal restriction enzymesand subcloned into the pcDNA3 vector which had been similarly digested.Following transformation into competent XL-1Blue host E. coli cells(Stratagene), individual colonies were grown up, plasmid extracted andthe presence of the crmA gene confirmed by both restriction mapping andDNA sequence analysis.

The resulting expression construct or pcDNA3 itself (as the control) wasintroduced into both MCF7 and BJAB cells by electroporation. MCF7 cellswere electroporated at 330 V, 960 μF in 0.4 cm cuvettes (BioRad), platedonto 100 mm dishes at varying dilutions and selected with G418 sulfate(Gibco-BRL) at a concentration of 500 μg/ml. After selection for threeweeks, pooled populations from each transfection were prepared bytrypsinizing dishes containing several hundred colonies. Additionally,clonal cells lines were derived by picking individual colonies fromselected dishes. BJAB cells were electroporated at 220 V, 960 μF in 0.4cm cuvettes (Bio-Rad) and selected in 3 mg/ml G418 sulfate. One dayfollowing transfection, a portion of the cell population was diluted ata concentration of 2500 cells/well in 96-well dishes from which clonallines were obtained after G418 selection. The remainder of the cellswere retained as the pooled population.

Experiment III

Cell Lines, TNF and Anti-Fas Antibody--The MCF7 cell line was aTNF-sensitive subclone obtained from Dr. David R. Spriggs (University ofWisconsin). MCF7 is a breast carcinoma epithelial cell line whichexpresses TNF receptor and is sensitive to TNF killing. The BJAB cellline was a gift of Dr. Fred Wang (Harvard). Recombinant TNF (specificactivity 6.27×10⁷ U/mg) was obtained from Genentech (South SanFrancisco, Calif.). Anti-Fas monoclonal antibody (clone CH-11, IgM) wasobtained from Pan Vera (Madison, Wis.).

RNA Isolation and Northern Analysis--RNA isolation and northern analysiswere carried out as described in Dixit, V. M. et al. (1990). J. BiolChem. 265:2973-2978. PCR (Perkin-Elmer) was used to generate a probespanning the coding region of the crmA gene as described above. β-actincDNA probe was purchased from Clontech (Palo Alto, Calif.) and thehybridization signal was visualized as a digitized image on a MolecularDynamics Phosphorimager.

Experiment IV

Induction of Apoptosis by TNF and anti-Fas--A subclone of the MCF7breast carcinoma epithelial cell line which expressed TNF receptor andwas sensitive to TNF killing was chosen for these studies. This cellline is characterized in Spriggs, D. R. et al. (1988) J. Clinc. Invest.,81:455-460. Further analysis revealed that Fas was also expressed onthese cells and that crosslinking with an anti-Fas monoclonal antibodyin the concomitant presence of the protein synthesis inhibitorcycloheximide induced cell death.

Cycloheximide aline for the duration of the assay did not induce celldeath beyond the negligible frequency of spontaneous apoptosis which isobserved in any untreated cell culture. Anti-Fas alone was notcytotoxic, but this is not surprising, since induction of cell death innon-lymphoid cells by Fas activation has been reported to require theconcomitant presence of either transcriptional or translationalinhibitors. See Itoh, N. et al. (1991) Cell 66:233-243.

A B-cell lymphoma cell line (BJAB) also was examined. It expresses ahigh level of Fas and was killed by the addition of anti-Fas antibody inthe absence of a protein synthesis inhibitor.

Cell death can occur by two biochemically and morphologically distinctprocesses: apoptosis and necrosis. In these studies, cell death wasfirst confirmed to be the result of TNF or anti-Fas induced apoptosis,not necrosis. Although various markers of apoptosis have been reported,the phenomenon is preferably defined at the morphological level and ischaracterized by chromatin condensation and margination along the innernuclear membrane, cytoplasmic condensation and membrane blebbing withoutdisintegration of the cellular membrane. See Duvall E. et al. (1986)Immunol. Today 7:115-119. Necrosis, conversely, is defined bycytoplasmic swelling and lysis of the cell membrane and, importantly,does not exhibit the chromatin margination characteristic of apoptosis.DNA laddering, representative of cleavage at internucleosomal intervals,is seen in some but not all forms of apoptosis, further emphasizing theimportance of morphological criteria in defining apoptosis. See Barres,B. A. et al. (1992) Cell 70:31-46. Nuclear morphology of cells dying inresponse to TNF or anti-Fas antibody was examined following stainingwith the DNA-binding dyes propidian iodine (MCF7 cells) and acridineorange (BJAB cells). Fluorescence microscopy laser scanning confocalmicroscopy demonstrated marked changes in nuclear morphology in the MCF7cells in response to either TNF or anti-Fas-CHX and in the BJAB cells inresponse to anti-Fas. Chromatin condensation was clearly visible byimmunofluorescence microscopy in both cell lines and formed the basisfor the later assays of apoptosis in transfected cell lines (FIG. 1B forBJAB). Confocal microscopy confirmed margination along the inner nuclearmembrane (FIGS. 1A and 1B inset). These morphological criteria ofapoptotic cell death were further confirmed by transmission electronmicroscopy. The MCF7 cells clearly demonstrated chromatin condensationand margination along the inner nuclear membrane, cytoplasmiccondensation and increased membrane blebbing in response to either TNFor anti-Fas+CHX (FIG. 1A). BJAB cells treated with anti-Fas antibodydemonstrated chromatin margination and cellular shrinkage typical ofapoptosis in lymphoid cells. Thus, both TNF and Fas induced genuineapoptotic cell death in these cell lines.

Experiment V

crmA Blocks TNF- and anti-Fas-Induced Apoptosis--To determine whethercrmA can function to inhibit cytokine-induced apoptosis, MCF7 and EJABcell lines were transfected with either the expression vector pcDNA3 byitself or as a crmA expression construct. Expression of the crmA genewas confirmed by northern analysis. Stable transfectants were generatedby neomycin selection, and pooled populations of neomycin-resistantcells were assayed for crmA expression. These pooled populations wereanalyzed for their sensitivity to TNF- and anti-Fas-induced apoptosis bydirect quantitation of apoptotic cells based on nuclear morphologyfollowing staining with DNA-binding dyes and visualization byfluorescence microscopy. Dramatic resistance was seen with either TNF orFas in both cell lines. This was remarkable, given that in the pooledpopulation of neomycin-resistant cells transfected with crmA, asignificant fraction of cells were likely not expressing crmA due to,among other reasons, nonproductive integration of the expressionconstruct into genomic DNA.

In addition to the pools, clonal lines were derived from both MCF7 andBJAB transfectants and challenged by activation of the TNF and Fas deathpathways. In the MCF-7 cell line, vector clones were uniformly sensitiveto apoptosis induced by either TNF or anti-Fas+CHX, whereas among thetransfected clones, those which expressed detectable crmA were totallyresistant to apoptosis (FIG. 3A). Indeed, lines expressing the highestlevels of crmA were totally resistant to apoptosis (FIG. 3A) and showedno morphologic cytopathic effects (FIG. 3B), demonstrating the crmA cancompletely block the TNF- and Fas-mediated death pathways. Similarly,among BJAB transfected clones, the crmA expressing the lines weremarkedly resistant to anti-Fas induced apoptosis whereas the vectorclones were universally sensitive (FIG. 4A). Importantly, in both MCF7and BJAB transfectants, those clones expressing the highest levels ofcrmA were the most resistant while those clones expressing little orundetectable levels were the most sensitive (FIG. 3A and FIG. 4A).Although direct visual quantitation of apoptotic nuclei is thepreferable measure of apoptosis, comparable results were obtained wheneither an MTT-conversion based death assay (FIG. 3A insert and FIG. 4Ainset) or crystal violet staining (FIG. 3C) was employed to assess cellsurvival.

The dose of death stimulus was increased to determine if protectionconferred by crmA from cytokine-induced apoptosis could be attenuated.Remarkably, crmA afforded comparably high levels of protection fromanti-Fas-induced apoptosis in response to doses of antibody 250 timesgreater than those needed to kill greater than 95% of the vectortransfected cells (FIG. 4B). Similar results were obtained when the doseof TNF was similarly varied for the MCF7 transfectants, implying thatcrmA is functioning as an exceptionally potent inhibitor of cell deathat a presumably critical step in the death pathway.

These results describe an important new function for crmA--the blockadeof TNF- and Fas-mediated apoptosis. Given the importance of both TNF andFas in the host anti-viral response, it is likely that this function ofcrmA is important for productive viral infection in vivo. crmArepresents yet another example of viral economy in which two importantfunctions, namely the inhibition of IL-1β production and the preventionof apoptosis, are embedded in one protein.

Additionally, this data have implications for the unification of deathpathways in general. First, the fact that crmA blocks both TNF- andFas-mediated apoptosis, especially in the MCF7 cells that possess bothreceptors, suggests that they signal death through a biochemicallycommon pathway. This hypothesis is supported by the finding that thecytoplasmic regions of both these receptors encompass a region ofhomology which has been defined by mutational analysis as a "deathdomain" and which presumably interacts with a common set of signaltransduction molecules. Further, it is not apparent that crmA blockscell death triggered by two very different stimuli: growth factorwithdrawal in neuronal cultures and, activation of cytokine receptors.It is of note that apoptosis in these two systems has been suggested tooccur through biochemically distinct pathways, in that apoptosis in theformer system is dependent on new protein synthesis and death is blockedby cycloheximide, whereas in contrast TNF- and Fas-mediated cytotoxicityis independent of new protein synthesis and is, in fact, enhanced bycycloheximide. Thus, at some point, the death pathway in both systemsconverges upon a crmA-inhibitable step, likely the activation of aprotease.

Throughout this application, various publications are referred to bytheir bibliographic citation. The disclosures of these references arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - - CACCGGAATT CCACCATGGA TATCTTCAGG GAAATCG      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:                        - - GCTCTAGACT CGAGTTAATT AGTTGTTGGA GAGCAATATC     - #                      - #    40                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1468 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 295..1317                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - - TCCATGGAAG AACGAAAGTA GTATAAAAGT AATAAAACAA AAAAAAGAAT AT -             #AAAAAATT     60                                                                 - - TATAGCCACT TTCTTTGAGG ACTGTTTTCC TGAAGGAAAT GAACCTCTGG AA -            #TTAGTTAG    120                                                                 - - ATATATAGAA TTAGTATACA CGCTAGATTA TTCTCAAACT CCTAATTATG AC -            #AGACTACG    180                                                                 - - TAGACTGTTT ATACAAGATT GAAAATATAT TTCTTTTTAT TGAGTGGTGG TA -            #GTTACGGA    240                                                                 - - TATCTAATAT TAATATTAGA CTATCTCTAT CGTCACACAA CAAAATCGAT TG - #CC        ATG      297                                                                                      - #                  - #                  - #             Met                                                                                               - #                  - #                  - #                - - GAT ATC TTC AGG GAA ATC GCA TCT TCT ATG AA - #A GGA GAG AAT GTA        TTC      345                                                                    Asp Ile Phe Arg Glu Ile Ala Ser Ser Met Ly - #s Gly Glu Asn Val Phe                        5    - #              10    - #              15                  - - ATT TCT CCA CCG TCA ATC TCG TCA GTA TTG AC - #A ATA CTG TAT TAT GGA          393                                                                       Ile Ser Pro Pro Ser Ile Ser Ser Val Leu Th - #r Ile Leu Tyr Tyr Gly                    20         - #         25         - #         30                      - - GCT AAT GGA TCC ACT GCT GAA CAG CTA TCA AA - #A TAT GTA GAA AAG GAG          441                                                                       Ala Asn Gly Ser Thr Ala Glu Gln Leu Ser Ly - #s Tyr Val Glu Lys Glu                35             - #     40             - #     45                          - - GCG GAC AAG AAT AAG GAT GAT ATC TCA TTC AA - #G TCC ATG AAT AAA GTA          489                                                                       Ala Asp Lys Asn Lys Asp Asp Ile Ser Phe Ly - #s Ser Met Asn Lys Val            50                 - # 55                 - # 60                 - # 65       - - TAT GGG CGA TAT TCT GCA GTG TTT AAA GAT TC - #C TTT TTG AGA AAA ATT          537                                                                       Tyr Gly Arg Tyr Ser Ala Val Phe Lys Asp Se - #r Phe Leu Arg Lys Ile                            70 - #                 75 - #                 80              - - GGA GAT AAT TTC CAA ACT GTT GAC TTC ACT GA - #T TGT CGC ACT GTA GAT          585                                                                       Gly Asp Asn Phe Gln Thr Val Asp Phe Thr As - #p Cys Arg Thr Val Asp                        85     - #             90     - #             95                  - - GCG ATC AAC AAG TGT GTT GAT ATC TTC ACT GA - #G GGG AAA ATT AAT CCA          633                                                                       Ala Ile Asn Lys Cys Val Asp Ile Phe Thr Gl - #u Gly Lys Ile Asn Pro                   100          - #       105          - #       110                      - - CTA TTG GAT GAA CCA TTG TCT CCA GAT ACC TG - #T CTC CTA GCA ATT AGT          681                                                                       Leu Leu Asp Glu Pro Leu Ser Pro Asp Thr Cy - #s Leu Leu Ala Ile Ser               115              - #   120              - #   125                          - - GCC GTA TAC TTT AAA GCA AAA TGG TTG ATG CC - #A TTT GAA AAG GAA TTT          729                                                                       Ala Val Tyr Phe Lys Ala Lys Trp Leu Met Pr - #o Phe Glu Lys Glu Phe           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - ACC AGT GAT TAT CCC TTT TAC GTA TCT CCA AC - #G GAA ATG GTA GAT        GTA      777                                                                    Thr Ser Asp Tyr Pro Phe Tyr Val Ser Pro Th - #r Glu Met Val Asp Val                          150  - #               155  - #               160              - - AGT ATG ATG TCT ATG TAC GGC GAG GCA TTT AA - #T CAC GCA TCT GTA AAA          825                                                                       Ser Met Met Ser Met Tyr Gly Glu Ala Phe As - #n His Ala Ser Val Lys                       165      - #           170      - #           175                  - - GAA TCA TTC GGC AAC TTT TCA ATC ATA GAA CT - #G CCA TAT GTT GGA GAT          873                                                                       Glu Ser Phe Gly Asn Phe Ser Ile Ile Glu Le - #u Pro Tyr Val Gly Asp                   180          - #       185          - #       190                      - - ACT AGT ATG GTG GTA ATT CTT CCA GAC AAT AT - #T GAT GGA CTA GAA TCC          921                                                                       Thr Ser Met Val Val Ile Leu Pro Asp Asn Il - #e Asp Gly Leu Glu Ser               195              - #   200              - #   205                          - - ATA GAA CAA AAT CTA ACA GAT ACA AAT TTT AA - #G AAA TGG TGT GAC TCT          969                                                                       Ile Glu Gln Asn Leu Thr Asp Thr Asn Phe Ly - #s Lys Trp Cys Asp Ser           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - ATG GAT GCT ATG TTT ATC GAT GTG CAC ATT CC - #C AAG TTT AAG GTA        ACA     1017                                                                    Met Asp Ala Met Phe Ile Asp Val His Ile Pr - #o Lys Phe Lys Val Thr                          230  - #               235  - #               240              - - GGC TCG TAT AAT CTG GTG GAT GCG CTA GTA AA - #G TTG GGA CTG ACA GAG         1065                                                                       Gly Ser Tyr Asn Leu Val Asp Ala Leu Val Ly - #s Leu Gly Leu Thr Glu                       245      - #           250      - #           255                  - - GTG TTC GGT TCA ACT GGA GAT TAT AGC AAT AT - #G TGT AAT TCA GAT GTG         1113                                                                       Val Phe Gly Ser Thr Gly Asp Tyr Ser Asn Me - #t Cys Asn Ser Asp Val                   260          - #       265          - #       270                      - - AGT GTC GAC GCT ATG ATC CAC AAA ACG TAT AT - #A GAT GTC AAT GAA GAG         1161                                                                       Ser Val Asp Ala Met Ile His Lys Thr Tyr Il - #e Asp Val Asn Glu Glu               275              - #   280              - #   285                          - - TAT ACA GAA GCA GCT GCA GCA ACT TGT GCG CT - #G GTG GCA GAC TGT GCA         1209                                                                       Tyr Thr Glu Ala Ala Ala Ala Thr Cys Ala Le - #u Val Ala Asp Cys Ala           290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - TCA ACA GTT ACA AAT GAG TTC TGT GCA GAT CA - #T CCG TTC ATC TAT        GTG     1257                                                                    Ser Thr Val Thr Asn Glu Phe Cys Ala Asp Hi - #s Pro Phe Ile Tyr Val                          310  - #               315  - #               320              - - ATT AGG CAT GTC GAT GGC AAA ATT CTT TTC GT - #T GGT AGA TAT TGC TCT         1305                                                                       Ile Arg His Val Asp Gly Lys Ile Leu Phe Va - #l Gly Arg Tyr Cys Ser                       325      - #           330      - #           335                  - - CCA ACA ACT AAT TAAATCACAT TCTTAATATT AGAATATTAG AA - #TATTATAT             1357                                                                       Pro Thr Thr Asn                                                                       340                                                                    - - AGTTAAGATT TTTACTAATT GGTTAACCAT TTTTTTAAAA AAATAGAAAA AA -             #AACATGTT   1417                                                                 - - ATATTAGCGA GGGTCGTTAT TCTTCCAATT GCAATTGGTA AGATGACGGC C - #               1468                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 341 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:4:                     - - Met Asp Ile Phe Arg Glu Ile Ala Ser Ser Me - #t Lys Gly Glu Asn Val        1               5 - #                 10 - #                 15              - - Phe Ile Ser Pro Pro Ser Ile Ser Ser Val Le - #u Thr Ile Leu Tyr Tyr                   20     - #             25     - #             30                  - - Gly Ala Asn Gly Ser Thr Ala Glu Gln Leu Se - #r Lys Tyr Val Glu Lys               35         - #         40         - #         45                      - - Glu Ala Asp Lys Asn Lys Asp Asp Ile Ser Ph - #e Lys Ser Met Asn Lys           50             - #     55             - #     60                          - - Val Tyr Gly Arg Tyr Ser Ala Val Phe Lys As - #p Ser Phe Leu Arg Lys       65                 - # 70                 - # 75                 - # 80       - - Ile Gly Asp Asn Phe Gln Thr Val Asp Phe Th - #r Asp Cys Arg Thr Val                       85 - #                 90 - #                 95              - - Asp Ala Ile Asn Lys Cys Val Asp Ile Phe Th - #r Glu Gly Lys Ile Asn                  100      - #           105      - #           110                  - - Pro Leu Leu Asp Glu Pro Leu Ser Pro Asp Th - #r Cys Leu Leu Ala Ile              115          - #       120          - #       125                      - - Ser Ala Val Tyr Phe Lys Ala Lys Trp Leu Me - #t Pro Phe Glu Lys Glu          130              - #   135              - #   140                          - - Phe Thr Ser Asp Tyr Pro Phe Tyr Val Ser Pr - #o Thr Glu Met Val Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Ser Met Met Ser Met Tyr Gly Glu Ala Ph - #e Asn His Ala Ser        Val                                                                                             165  - #               170  - #               175             - - Lys Glu Ser Phe Gly Asn Phe Ser Ile Ile Gl - #u Leu Pro Tyr Val Gly                  180      - #           185      - #           190                  - - Asp Thr Ser Met Val Val Ile Leu Pro Asp As - #n Ile Asp Gly Leu Glu              195          - #       200          - #       205                      - - Ser Ile Glu Gln Asn Leu Thr Asp Thr Asn Ph - #e Lys Lys Trp Cys Asp          210              - #   215              - #   220                          - - Ser Met Asp Ala Met Phe Ile Asp Val His Il - #e Pro Lys Phe Lys Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Gly Ser Tyr Asn Leu Val Asp Ala Leu Va - #l Lys Leu Gly Leu        Thr                                                                                             245  - #               250  - #               255             - - Glu Val Phe Gly Ser Thr Gly Asp Tyr Ser As - #n Met Cys Asn Ser Asp                  260      - #           265      - #           270                  - - Val Ser Val Asp Ala Met Ile His Lys Thr Ty - #r Ile Asp Val Asn Glu              275          - #       280          - #       285                      - - Glu Tyr Thr Glu Ala Ala Ala Ala Thr Cys Al - #a Leu Val Ala Asp Cys          290              - #   295              - #   300                          - - Ala Ser Thr Val Thr Asn Glu Phe Cys Ala As - #p His Pro Phe Ile Tyr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Ile Arg His Val Asp Gly Lys Ile Leu Ph - #e Val Gly Arg Tyr        Cys                                                                                             325  - #               330  - #               335             - - Ser Pro Thr Thr Asn                                                                  340                                                              __________________________________________________________________________

What is claimed is:
 1. A method for preventing or inhibiting apoptosis,in a mammalian cell, wherein the cell is induced to apoptosis by thebinding of a ligand to its cell surface receptor, comprising introducinginto the mammalian cell an effective amount of a nucleic acid moleculecoding for a gene product having crmA biological activity and underconditions such that apoptosis is prevented or inhibited.
 2. The methodof claim 1, wherein the mammalian cell has a receptor selected from thegroup consisting of a Fas receptor, a TNF receptor, a TCR, a CD4receptor and a CD3 receptor.
 3. The method of claim 2, wherein the cellis a CD4⁺ T cell.
 4. The method of claim 3, wherein the CD4⁺ T cellcontains a human immunodeficiency virus (HIV).
 5. The method of claim 1,wherein the nucleic acid molecule is introduced into the mammalian cellin vitro.
 6. The method of claim 1, wherein the nucleic acid molecule isintroduced into the mammalian cell ex vivo.
 7. The method of claim 1,wherein the nucleic acid molecule is introduced into the mammalian cellin vivo.
 8. The method of claim 1, wherein the nucleic acid comprisesDNA encoding poxvirus crmA or a DNA encoding a biologically activefragment of poxvirus crmA.
 9. A method for maintaining T cell viabilityin a subject infected with the human immunodeficiency virus, comprisingadministering to the subject an effective amount of a nucleic acidmolecule coding for a gene product having crmA biological activity andunder conditions such that T cells remain viable.
 10. The method ofclaim 9, wherein the subject is an animal.
 11. The method of claim 9,wherein the subject is a human patient.